Kountz P D, el-Maghrabi M R, Pilkis S J
Arch Biochem Biophys. 1985 May 1;238(2):531-43. doi: 10.1016/0003-9861(85)90197-3.
6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were copurified to homogeneity from bovine liver. The purification scheme consisted of polyethylene glycol precipitation, anion-exchange and Blue-Sepharose chromatography, substrate elution from phosphocellulose, and gel filtration. The bifunctional enzyme had an apparent molecular weight of 102,000 and consisted of two subunits (Mr 49,000). The kinase had a Km for ATP of 12 microM and a S0.5 for fructose 6-phosphate of 150 microM while the bisphosphatase had a Km for fructose 2,6-bisphosphate of 7 microM. Both activities were subject to modulation by various effectors. Inorganic phosphate stimulated both activities, while alpha-glycerolphosphate inhibited the kinase and stimulated the bisphosphatase. The pH optimum for the 6-phosphofructo-2-kinase activity was 8.5, while the fructose-2,6-bisphosphatase reaction was maximal at pH 6.5. Incubation of the purified enzyme with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in 32P incorporation to the extent of 0.7 mol/mol enzyme subunit with concomitant inhibition of the kinase activity and activation of the bisphosphatase activity. The mediation of the bisphosphatase reaction by a phosphoenzyme intermediate was suggested by the isolation of a stable labeled phosphoenzyme when the enzyme was incubated with fructose 2,6-[2-32P]bisphosphate. The pH dependence of hydrolysis of the phospho group suggested that it was linked to the N3 of a histidyl residue. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver has properties essentially identical to those of the rat liver enzyme, suggesting that hepatic fructose 2,6-bisphosphate metabolism is under the same control in both species.
6-磷酸果糖-2-激酶和果糖-2,6-双磷酸酶活性从牛肝中共纯化至均一。纯化方案包括聚乙二醇沉淀、阴离子交换和蓝葡聚糖层析、从磷酸纤维素上进行底物洗脱以及凝胶过滤。该双功能酶的表观分子量为102,000，由两个亚基（Mr 49,000）组成。激酶对ATP的Km为12 microM，对6-磷酸果糖的S0.5为150 microM，而双磷酸酶对果糖-2,6-双磷酸的Km为7 microM。两种活性均受多种效应物的调节。无机磷酸盐刺激两种活性，而α-甘油磷酸抑制激酶并刺激双磷酸酶。6-磷酸果糖-2-激酶活性的最适pH为8.5，而果糖-2,6-双磷酸酶反应在pH 6.5时最大。将纯化的酶与[γ-32P]ATP和cAMP依赖性蛋白激酶的催化亚基一起孵育，导致32P掺入量达到0.7 mol/mol酶亚基，同时激酶活性受到抑制，双磷酸酶活性被激活。当酶与果糖2,6-[2-32P]双磷酸一起孵育时，通过分离稳定的标记磷酸酶中间体，提示双磷酸酶反应由磷酸酶中间体介导。磷酸基团水解的pH依赖性表明它与组氨酸残基的N3相连。牛肝中的6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶的性质与大鼠肝酶的性质基本相同，表明两种物种的肝脏果糖-2,6-双磷酸代谢受相同的调控。
